It is well know that transposons are a class of DNA sequences that can move from an episome to a chromosomal site or from one chromosomal site to another. Transposons are known in both prokaryotes, such as bacteria, as well as in eukaryotes, although there have been few transposons isolated from filamentous fungi.
Several groups have looked for transposons in filamentous fungi. The element pogo, which exists in multiple copies and at different sites in different strains of Neurospora crassa, was described by Schectman (1) and is believed to be a transposon. To date the most characterized transposon in filamentous fungi is Tad. Tad was isolated as a spontaneous mutant in the am (glutamate dehydrogenase) gene in an Adiopodoume strain of N. crassa isolated from the Ivory Coast. To detect mutations caused by insertion of a transposable element, Kinsey and Helber (2) isolated genomic DNA from 33 am mutant strains which were then screened by Southern analysis for restriction fragment size alterations. In two of the mutant strains, the mutation was shown to be caused by the insertion of a 7 kb element (Tad) into the am gene. Subsequently Kinsey (3) demonstrated that Tad was able to transpose between nuclei of heterokaryons, confirming that Tad was a retrotransposon and that there was a cytoplasmic phase involved in the retrotransposition events. More recently, Cambareri et al. (4) demonstrated that Tad was a LINE-like DNA element with two major open reading frames (ORFs) on the plus strand. Typical of LINE-like elements, Tad had no terminal repeats. Attempts to isolate mobile transposons in laboratory strains of N. crassa were unsuccessful.
A second retrotransposon was cloned by McHale et al. (5), who reported the isolation of CfT-1, an LTR-retrotransposon from Cladosporium fulvum. This transposon was 6968 bp in length and bounded by identical long terminal repeats of 427 bp, a 5 bp target site duplication. Virus-like particles were detected which co-sediment with reverse transcriptase activity in homogenates of this fungus.
Daboussi et al. (6) were the first to successfully use the niaD (nitrate reductase) gene as a transposon trap. The niaD mutants can be isolated by a direct selection for chlorate resistance (7). The strategy employed was to isolate niaD mutants amongst six isolates belonging to different races of the fungus Fusarium oxysporum. More than 100 niaD mutants were isolated from each isolate and examined for instability. One strain, F24, yielded up to 10% unstable niaD mutants. Assuming that the genetic instability of the niaD mutants was caused by transposable elements, it seemed plausible that this isolate contained mobile transposons. A stable niaD mutant in the F24 was transformed with the cloned niaD gene from A. nidulans because the F. oxysporum niaD gene had not been cloned. Unstable niaD mutants were isolated in transformants containing the A. nidulans niaD gene. Two unstable niaD mutants were shown by Southern blot analysis to contain a insertion of 1.9 kb in size. Analysis of this element, Fot1, revealed it was 1928 bp long, had a 44 bp inverted terminal repeats, contained a large open reading frame, and was flanked by a 2 bp (TA) target site duplication. Very recently, Daboussi et al. (8) have reported the cloning of a new transposable element from an unstable niaD mutant. This element, FML (Fusarium mariner-like), is 1280 bp long and has inverted repeats of 27 bp. The FML element inserts into a TA site and excises imprecisely.
Using the characterization of unstable niaD mutants strategy, Lebrun et al. (9) were able to isolate a transposon from Magnaporthe grisea. However, in this case the A. nidulans niaD gene which was transformed into M. grisea by transformation was used as a transposon trap. The element inserted into the niaD gene was shown to belong to a family of M. grisea LTR-retrotransposons, Fos 1 (Schull and Hamer, unpublished) and Mag1 (Farman and Leong, unpublished). The cloned retro-element was 5.6 kb and the target site (ATATT) was shown to be duplicated. All revertants from this mutant examined had one copy of the LTR left at the point of insertion. A second transposon, Pot2, from M. grisea was recently cloned by Kachroo et al. (10). The strategy used to clone Pot2 was to analyze the fingerprint patterns of repetitive DNA's which were cloned from the M. grisea genome. A repetitive family present in both rice and non-rice pathogens of M. grisea in high copy number was cloned. The element, 1857 bp in size, has a 43 bp perfect terminal inverted repeats (TIR) and 16 bp direct repeats within the TIRs. An open reading frame was shown to display extensive identity to that of Fot1 of F. oxysporum. As with Fot1, the Pot2 element duplicates the dinucleotide TA at the target insertion site. Pot2 was shown to be present at a copy number of approximately 100 per haploid genome.
Several groups have reported looking without success for transposons in laboratory strains of A. nidulans (Kinghorn personnel communication, 5). One explanation for the lack of transposons in laboratory strains is that the desirable features of strain stability required for genetic analysis may preclude strains with mobile transposon. By using the niaD gene as a transposon trap we have identified and isolated a transposable element from the industrially important fungus A. niger var. awamori. This element, Vader, is present in approximately 15 copies in A. niger and A. niger var. awamori. Southern analysis of A. nidulans with this element indicates that this transposable element was absent from one laboratory strain and only present as a single copy in a second laboratory strain. These results support the notion that laboratory strains of A. nidulans contain very few transposons.